Estrogen loss increases osteoclastogenesis, decreases osteoclast and increases osteoblast apoptosis, and causes loss of bone by decreasing anti-oxidant defense in these cell types. In addition, an age-dependent decrease in bone strength and mass in ovary-intact C57BL/6 mice is temporally associated with an agedependent increase in the prevalence of osteoblast apoptosis, decreased glutathione reductase (GSR) activity and corresponding increases in the production of reactive oxygen species (ROS) in the bone marrow and the phosphorylation of p66shc in vertebrae. Based on this evidence, it is hypothesized that increased ROS levels with advancing age is a fundamental mechanism of the age-dependent decline of bone strength and mass, and loss of estrogens exaggerates the adverse effects of aging on bone by decreasing defense against ROS, thereby, contributing perpetually to the loss of bone mass and strength that persists for decades after menopause, and is associated with old age. Estrogens enhance defense against ROS by modulating the activity of anti-oxidant enzymes, attenuating ROS-mediated induction of cytokines, and modulating signaling cascades that have been implicated in the defense against oxidative stress, apoptosis, and aging. All these effects are mediated by extranuclear (nongenotropic) actions of the ER on cytoplasmic kinases and result in changes in the birth and lifespan of osteoblasts, osteoclasts, and osteocytes. To test these interrelated hypotheses, in vitro studies are proposed to investigate whether the effects of estrogens on osteoblast and osteoclast apoptosis, as well as on osteoclastogenesis, are, at least in part, a direct consequence of the ability of these hormones to: a) antagonize the actions of ROS by increasing the levels of glutathione;and/or b) counteract ROS-activated signals on cytoplasmic kinases, and/or downstream transcription factors and the resulting upregulation of cytokines. Further, the role of oxidative stress in the molecular and cellular mechanism responsible for the loss of bone strength and mass associated with aging and the extent to which estrogen deficiency and the resulting attenuation of defense against oxidative stress in osteoblasts or osteoclasts contributes to these changes will be studied in mice over-expressing GSR in osteoblasts or osteoclasts. Finally, mice in which ERa will be deleted from osteoblasts or osteoclasts by means of CreXLoxP recombination;mice heterozygotes for an ERa knock-in mutant which cannot bind to ERE;and mice over-expressing an ERa mutant, which is incapable of membrane localization and prevents kinase-mediated signaling of the endogenous receptor, will be used to examine whether the antioxidant actions of estrogens on osteoblasts and osteoclasts in vivo are ER-dependent and nongenotropic. This work should help us understand how women become increasingly more susceptible to fractures as they grow old. Moreover, it mav lead to the discovery of more effective treatments or even a cure for osteoporosis.